Quality Control

Biological Activity

LDN-193189 is a highly potent small molecule inhibitor of bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 with IC50 values of 5 nM and 30 nM, respectively. BMPs induce Smad1/5/8 plus non-Smad pathways, such as MAPK and Akt. BMP signaling pathway is the key regulators of cell fate decisions during embryogenesis and tissue homeostasis. LDN-193189 inhibits ectopic ossification and a potential agent in the treatment of NSCLC lung tumors, showing significant in vivo clinical utility. In addition, it inhibits the activation of p38, ERK1/2 and Akt in C2C12 cells. LDN-193189 only weakly inhibits ALK4, ALK5, and ALK7. LDN-193189 affects not only the Smad but also the non-Smad signalling pathways induced by either BMP2, BMP6 or GDF5.

Customer Product Validations & Biological Datas

Source

J Biol Chem (2015). Figure 4. LDN-193189

Method

immunoblotting

Cell Lines

C2C12 cells

Concentrations

5 μM

Incubation Time

30 min

Results

Pretreatment with dorsomorphin or LDN-193189 led to reduced levels of phosphorylated Smad2/3 following GDF8 stimulation (Fig. 4A), which were comparable with their effects on BMP2-induced phosphorylation of Smad1/5 (Fig. 4B), whereas there was no effect on TGF- induced Smad2/3 signals (Fig. 4A).

Source

J Biol Chem (2015). Figure 2. LDN-193189

Method

immunoblotting

Cell Lines

C2C12 cells

Concentrations

0.5 μM

Incubation Time

30 min

Results

A similar effect of dorsomorphin and LDN-193189 treatment was observed in undifferentiated primary human myoblasts as well as in the mouse myoblast cell line C2C12

Protocol

Cell Experiment

Cell lines

C2C12 cells

Preparation method

Alkaline phosphatase activity We seeded C2C12 cells into 96-well plates at 2,000 cells per well in DMEM supplemented with 2% FBS. We treated the wells in quadruplicate with BMP ligands and LDN-193189 or vehicle. We collected the cells after 6 d in culture in 50 μl Tris-buffered saline and 1% Triton X-100. We added the lysates to p-nitro-phenylphosphate reagent in 96-well plates (Sigma) for 1 h and then evaluated alkaline phosphatase activity (absorbance at 405 nm). We measured cell viability and quantity by Cell Titer Aqueous One (absorbance at 490 nm, Promega), using replicate wells treated identically to those used for alkaline phosphatase measurements.

Concentrations

0,2,8,31,125,500nM

Incubation time

6 days

Animal Experiment

Animal models

C57BL/6 mouse FOP model

Formulation

unknown

Dosages

3 mg /kg every 12 h

Administration

intraperitoneally

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species

Mouse

Rat

Rabbit

Guinea pig

Hamster

Dog

Weight (kg)

0.02

0.15

1.8

0.4

0.08

10

Body Surface Area (m2)

0.007

0.025

0.15

0.05

0.02

0.5

Km factor

3

6

12

8

5

20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by

Animal B Km

Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.